Nitrosylation of protein SH groups and amino acid residues as a therapeutic modality

ABSTRACT

Nitrosylation of proteins and amino acid groups enables selective regulation of protein function, and also endows the proteins and amino acids with additional smooth muscle relaxant and platelet inhibitory capabilities. Thus, the invention relates to novel compounds achieved by nitrosylation of protein thiols. Such compounds include: S-nitroso-t-PA, S-nitroso-cathepsin; S-nitroso-lipoprotein; and S-nitroso-immunoglobulin. The invention also relates to therapeutic use of S-nitroso-protein compounds for regulating protein function, cellular metabolism and effecting vasodilation, platelet inhibition, relaxation of non-vascular smooth muscle, and increasing blood oxygen transport by hemoglobin and myoglobin. The compounds are also used to deliver nitric oxide in its most bioactive form in order to achieve the effects described above, or for in vitro nitrosylation of molecules present in the body. The invention also relates to the nitrosylation of oxygen, carbon and nitrogen moieties present on proteins and amino acids, and the use thereof to achieve the above physiological effects.

This invention was made with government support under RO1-HL40411,HL43344 and RR04870, awarded by The National Institutes of Health. Thegovernment has certain rights in the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. Ser. No. 08/198,854filed on Feb. 17, 1994, which is a divisional application of U.S. Ser.No. 07/943,835, filed Sep. 14, 1992, now abandoned, which is acontinuation-in-part of U.S. Ser. No. 07/791,668, filed Nov. 14, 1991(abandoned).

BACKGROUND OF THE INVENTION FIELD OF THE INVENTION

This invention relates to nitrosylation of proteins and amino acids as atherapeutic modality. In particular, the invention relates toS-nitroso-protein compounds and their use as a means to selectivelyregulate specific protein functions, to selectively regulate cellularfunction, to endow the protein with new smooth muscle relaxant andplatelet inhibitory properties and to provide targeted delivery ofnitric oxide to specific bodily sites.

Additionally, the invention relates to nitrosylation of additional sitessuch as oxygen, carbon and nitrogen, present on proteins and aminoacids, as a means to achieve the above physiological effects. Thetherapeutic effects my be achieved by the administration of nitrosylatedproteins and amino acids as pharmaceutical compositions, or bynitrosylation of proteins and amino acids in vivo through theadministration of a nitrosylating agent, perhaps in the form of apharmaceutical composition.

BRIEF DESCRIPTION OF THE BACKGROUND ART

The reaction between low molecular weight thiols, such as cysteine,homoeysteine, and N-acetyleysteine, and nitric oxide (NO) has beenstudied in biological system. NO has been shown to induce relaxation ofvascular smooth muscle, and inhibition of platelet aggregation, throughactivation of guanylate eyclase and elevation of eyelie GMP levels.Evidence exists that low molecular weight thiols react readily with NOto form S-nitrosothiols, which are significantly more stable than NOitself, and act as potent vasodilators and platelet inhibitors. Theseadduets have also been proposed as biologically active intermediates inthe metabolism of organic nitrates (Ignarro et al., J. Pharmacol. Exp.Ther. 218:739 (1981); Mellion, et al., Mol. Pharmacol. 23:653 (1983);Loscalso, et al, J. Clin. Invest. 76:966 (1985)).

Many proteins of physiological significance possess intramolecularthiois in the form of cysteine residues. These thiol groups are ofcritical importance in the functional properties of such proteins. Thesesulfhydryl groups are highly specialized and utilized extensively inphysiological processes such as metabolic regulation, structuralstabilization, transfer of reducing equivalents, detoxification pathwaysand enzyme catalysis (Gilbert, H. F., "Molecular and Cellular Aspects ofThiol-Disulfide Exchange", Advances in Enzymology, A. Miester, J. Wiley& Sons, Eds. New York 1990, pages 69-172.)

Thiols are also present on those proteins the function of which is totransport and deliver specific molecules to particular bodily tissues.For example, lipoproteins are globular particles of high molecularweight that transport nonpolar lipids through the plasma. These proteinscontain thiols in the region of the protein which controls cellularuptake of the lipoprotein (Mahley et al. JAMA 265:78-83 (1991)).Hyper-liproteinemias, resulting from excessive lipoprotein (and thus,lipid) uptake, cause life-threatening diseases such as astherosclerosisand pancreatitis.

The thiol contained in hemoglobin regulates the affinity of hemoglobinfor oxygen, and thus has a critical role in the delivery of oxygen tobodily tissues. The reaction between the free NO radical occurs at theiron-binding site of hemoglobin, and not the thiol. As a result,methemoglobin is generated, which impairs oxygen-hemoglobin binding, andthus, oxygen transport. Other proteins such as thrombolytic agents,immunoglobulins, and albumin, possess free thiol groups that areimportant in regulating protein function.

Protein thiols may, under certain pathophysiological conditions, cause aprotein to exert a detrimental effect. For example, cathepsin, asulfhydryl enzyme involved in the breakdown of cellular constituents, iscritically dependent upon sulfhydryl groups for proteolytic activity.However, uncontrolled proteolysis caused by this enzyme leads to timedamage; specifically lung damage caused by smoking.

The reaction between NO and the thiols of intact protein molecules haspreviously been studied only to a very limited extent. There is someevidence for the reaction between proteins and nitro(so)-containingcompounds in vivo. Investigators have observed that the denitrificationof nitroglycerin in plasma is catalyzed by the thiol of albumin (Chonget al., Drug Met. and Disp. 18:61 (1990), and these authors suggest ananalogy between this mechanism and the thiol-dependent enzymaticdenitrification of nitroglycerin with glutathione S-transferase in areaction which generates tionitrates (Keene et al., JBC 251:6183(1976)). In addition, hemoproteins have been shown to catalyzedenitrification of nitroglycerin, and to react by way of thiol groupswith certain nitroso-compounds as pan of the hypothesized detoxificationpathway for arylhydroxylamines (Bennett et al., J. Pharmacol. Exp. Ther.237:629 (1986); Umemoto et al., Biochem. Biophys. Res. Commun. 151:1326(1988)). The chemical identity of intermediates in these reactions isnot known.

Nitrosylation of amino acids can also be accomplished at sites otherthan the thiol group. Tyrosine, an aromatic amino acid, which isprevalent in proteins, peptides, and other chemical compounds, containsa phenolic ring, hydroxyl group, and amino group. It is generally knownthat nitration of phenol yields ortho-nitrophenyl and para-nitrophenylC-nitrosylation products. Nitrosylation of tyrosine, using nitrous acid,has been shown to yield C-nitrosylated tyrosine (Reeve, R. M.,Histrochem. Cytochem. 16(3): 191-8 (1968)), and it has been suggestedthat this process produces O-nitro-tyrosine as a preliminary productwhich then rearranges into the C-nitrosylated product. (Baliga, B. T.Org. Chem. 35(6): 2031-2032(1970); Bonnett et al., J. C. S. PerkinTrans. I; 2261-2264 (1975)).

The chemistry of amino acid side chains, such as those found on tyrosineand other aromatic amino acids, has a critical role in ensuring properenzymatic function within the body. In addition, the hydroxyl group oftyrosine plays a central role in a variety of cell regulatory functions,with phosphorylation of tyrosine being one such critical cell regulatoryevent. In addition to possessing bioactive side chains, these aromaticamino acids serve as precursors to numerous important biomolecules suchas hormones, vitamins, conezymes, and neurotransmitters.

The current state of the art lacks chemical methods for modifying theactivity and regulating the intermediary cellular metabolism of theamino acids and proteins which play a critical role in biologicalsystems. Moreover, the ability to regulate protein function bynitrosylation was, prior to the present invention, unappreciated in theart.

It is appreciated in the art that, as a result of their increasedmolecular weight and tertiary structure, protein molecules differsignificantly from low molecular weight thiols. Furthermore, because ofthese differences, it would not be expected that protein thiols could besuccessfully nitrosylated in the stone manner as low molecular weightthiols, or that, if nitrosylated, they would react in the same manner.Furthermore, it would be equally unexpected that nitrosylation ofadditional sites such as oxygen, carbon and nitrogen would provide ameans for regulation of protein function.

Because of the great importance of diverse proteins and amino acids inall biological systems, it would be extremely desirable to have a methodfor achieving selective regulation of protein and amino acid function.There are virtually unlimited situations in which the ability toregulate amino acid or protein function by nitrosylation would be oftremendous therapeutic significance. Examples of ways in whichregulation or modification of function could be achieved would be thefollowing: (1) To enhance or prolong the beneficial properties of theprotein or amino acid; (2) to imbue the protein or amino acid withadditional beneficial properties; (3) to eliminate detrimentalproperties of a protein or amino acid; and (4) to alter the metabolismor uptake of proteins or amino acids in physiological systems.

The present invention represents a novel method for achieving thesetherapeutically significant objectives by regulation of protein andamino acid function with either of the following methods: (1)administration of particular nitrosylated proteins or amino acids to apatient; and (2) nitrosylation of a protein or amino acid in vivo by theadministration of a nitrosylating agent to a patient. In addition, theinvention represents the discovery of exemplary S-nitroso-proteins andamino acids of great biological and pharmacological utility.

SUMMARY OF THE INVENTION

This invention is based on the discovery by the inventors thatnitrosylating thiols, as well as oxygen, carbon and nitrogen present onproteins and amino acids provides a means for achieving selectiveregulation of protein and amino acid function. This concept can beemployed to generate S-nitroso-protein compounds, as well as othernitrosylated proteins and amino acids, which possess specificproperties, and can be directly administered to a patient. In thealternative, the invention provides a means for in vivo regulation ofprotein or amino acid function by nitrosylation. The invention istherefore directed to novel. S-nitroso-proteins and the therapeutic usesthereof, as well as the nitrosylation of proteins in vivo, as atherapeutic modality. The invention is also directed to nitrosylation ofoxygen, carbon and nitrogen sites of proteins and amino acids, as atherapeutic modality. In particular, this invention is directed tocompounds comprising an S-nitroso-enzyme. Enzymes contained in thiscompound include time-type plasminogen activator, streptokinase,urokinase and cathepsin.

This invention is also directed to compounds comprisingS-nitroso-lipoprotein. Lipoproteins which may be contained in thecompound include chylomicrons, chylomicron remnant particles, verylow-density lipoprotein (VLDL), intermediate-density lipoprotein (IDL),low-density lipoprotein (LDL) high-density lipoprotein (HLD) andlipoprotein (a).

This invention is also directed to compounds comprisingS-nitroso-immunoglobulin. Immunoglobulins contained in this compoundinclude IgG, IgM, IgA, IgD, IgE.

The invention is also directed to the compound S-nitroso-hemoglobin.

The invention is also directed to the compound S-nitroso-myoglobin.

The invention is also directed to pharmaceutical compositions containingthe compounds of the invention, together with a pharmaceuticallyacceptable carrier.

The invention is also direct to a method for regulating oxygen deliveryto bodily sites by administering pharmaceutical compositions containingS-nitroso-hemoglobin and S-nitroso-myoglobin.

The invention also relates to methods for effecting vasodilation,platelet inhibition, and thrombolysis; and for treating cardiovasculardisorders, comprising administering the pharmaceutical compositions ofthe invention to an animal.

This invention is also directed to a method for effecting plateletinhibition, comprising administering a pharmaceutical compositioncomprised of S-nitroso-albumin. An additional embodiment of theinvention comprises the method for causing relaxation of airway smoothmuscle and for the treatment or prevention of respiratory disorders,comprising administering a pharmaceutical composition containingS-nitroso-albumin.

This invention also is directed to a method for causing vasodilation,platelet inhibition and thrombolysis, comprising administering anitrosylating agent to an animal.

This invention also is directed to a method for regulation of proteinfunction in vivo, comprising administering a nitrosylating agent to ananimal.

The invention is directed to a method for preventing the uptake of aprotein by cells, comprising administering a nitrosylating agent to apatient.

The invention is also directed to a method for causing relaxation ofnon-vascular smooth muscle, comprising administering the pharmaceuticalcompositions of the invention to an animal.

The invention is also directed to a method for regulating the functionof proteins in which the thiol is bound to a methyl group, comprisingthe steps of removing the methyl groups from the protein by selectivede-methylation, and reacting the free thiol group with a nitrosylatingagent.

The invention is also directed to a method for regulating the functionof a protein which lacks a free thiol group, comprising the steps ofadding a thiol group to the protein, and reacting the thiol group with anitrosylating agent.

The invention is also directed to a method for regulating cellularfunction, comprising the S-nitrosylation of a protein which is cellularcomponent or which affects cellular function.

The invention is also directed to a method for delivering nitric oxideto specific, targeted sites in the body comprising administering aneffective mount of the pharmaceutical compositions of the invention toan animal.

The invention is also directed to a method for inhibiting plateletfunction, comprising the nitrosylation of a protein of amino acid atother sites, in addition to thiol groups, which are present on saidprotein or amino acid.

The invention is also directed to a method for causing vasodilation,comprising the nitrosylation of a protein or amino acid at other sites,in addition to thiol groups, which are present on said protein or aminoacid.

The invention is also directed to a method for relaxing smooth muscle,comprising the nitrosylation of a protein or amino acid at other sites,in addition to thiol groups, which are present on said protein or aminoacid.

The invention is also directed to a method for regulating cellularfunction, comprising the nitrosylation of a protein or amino acid atother sites, in addition to thiol groups, which are present on saidprotein or amino acid.

The invention is also directed to a method for delivery of nitric oxideto specific, targeted sites in the body, comprising the nitrosylation ofa protein or amino acid at other sites, in addition to thiol groups,which are present on said protein or amino acid.

The sites which are nitrosylated are selected from the group consistingof oxygen, carbon and nitrogen.

The invention is also directed to a method for inhibiting plateletfunction, comprising administering a pharmaceutical compositioncomprised of a compound selected from the group consisting of anyS-nitroso-protein.

The invention is also directed to a method for causing vasodilation,comprising administering a pharmaceutical composition comprised of acompound selected from the group consisting of any S-nitroso-protein.

The invention is also directed to a method for treatment or preventionof cardiovascular disorders, comprising administering a pharmaceuticalcomposition comprised of a compound selected from the group consistingof any S-nitroso-protein.

The invention is directed to a method for relaxing non-vascular smoothmuscle, comprising administering a pharmaceutical composition comprisedof a compound selected from the group consisting of anyS-nitroso-protein.

The invention is also directed to a method for treatment or preventionof respiratory disorders, comprising administering a pharmaceuticalcomposition comprised of a compound selected from the group consistingof any S-nitroso-protein.

The invention is also directed to a method for delivering nitric oxideto specific, targeted sites in the body, comprising administering apharmaceutical composition comprised of a compound selected from thegroup consisting of any S-nitroso-protein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. S--NO-t-PA spectroscopy.

1a: The ultraviolet absorption spectrum S--NO-t-PA (15 μM) relative tounmodified t-PA.

1b: The chemical shift of S-[¹⁵ N])O-t-PA (35 μM) at 751 ppm relative tonitrite using [¹⁵ N]NMR.

FIG. 2. Determination of S--NO bond formation in the synthesis ofS--NO-t-PA.

FIG. 3. [¹⁵ N]-NMR Spectrum of [¹⁵ N]-labeled S-nitroso-BSA.

FIG. 4. Concentration-dependent binding of t-PA and S--NO-t-PA tofibrinogen-coated wells.

FIG. 5. Double reciprocal plots for S--NO-t-PA. Results are expressed asmean ±S.D. (n=3).

5a: Double reciprocal plot 1/v versus 1/s for t-PA and S--NO-t-PAgenerated against the chromogenic substrate S2288.

5b: The curves for activation of glu-plasminogen (0.1-10 μM) by t-PA andS--NO-t-PA, generated using the plasmin-specific chromogenic substrateS2251.

FIG. 6. Fibrogen stimulation of enzymatic activity of t-PA (clear bars)and S--NO-t-PA (hatched bars), compared in the coupled enzyme assay atconcentrations of 0.1 μM and 1.0 μM of plasminogen.

FIG. 7. Increases in intracellular platelet cyclic GMP, caused byS--NO-t-PA.

FIG. 8. Inhibition of platelet aggregation by S--NO-t-PA.

FIG. 9. Comparison of S--NO-t-PA-induced vasorelazation caused by (a)t-PA (150 nM), (b) S--NO-t-PA (150 nM), and (c) S--NO-t-PA (150 nM).

FIG. 10. Dog-dependent relaxation of vascular smooth muscle andinhibition of platelet aggregation caused by S-nitroso-BSA (S--NO-BSA).

FIG. 11. Representative tracings of vessel relaxation and plateletinhibition caused by S-nitroso-BSA (S--NO-BSA).

11a: Illustrative tracings comparing the platelet inhibitory effects of(a) S--NO-BSA; (b) NaNO₂ ; (c) BSA; (d) iodoacetamide-treated BSAexposed to NO generated from acidified NaNO₂.

11b: Illustrative tracings comparing the vasodilatory effects of (a) BSA(1.4 μM); (b) iodoacetamide-treated BSA treated with NO generated fromacidified NaNO₂ as described in FIG. 3a; (c) S--NO-BSA (1.4 μM) afterplatelets were pretreated with 1 μM methylene blue for ten minutes; (d)S--NO-BSA (1.4 μM).

FIG. 12. Coronary blood flow in anesthetized dogs, following infusion ofS-nitroso-BSA.

FIG. 13. Duration of increased coronary blood flow, following infusionof S-nitroso-BSA.

FIG. 14. Coronary vasodilation, following infusion of S-nitroso-BSA.

FIG. 15. Dose-dependent vasodilatory response caused byS-nitroso-cathepsin.

FIG. 16. Tracings of dose-dependent inhibition of platelet aggregationcaused by S-nitroso-LDL.

FIG. 17. Representative tracings of vessel relaxation caused byS-nitroso-LDL.

FIG. 18. Tracings of dose-dependent inhibition of platelet aggregationcaused by S-nitroso-immunoglobulin.

FIG. 19. Representative tracings of vessel relaxation caused byS-nitroso-immunoglobulin.

FIG. 20. Concentration-dependent relaxation of airway smooth musclecaused by S--NO-BSA.

FIG. 21. Nitrosylation of L-tyrosine.

21a: [¹⁵ N]-NMR spectrum.

21b: [¹ H]-NMR spectrum.

21c: FTIR spectrum

21d: UV spectrum of 1.8 mM of tyrosine.

21e: UV spectrum for 34 mM of tyrosine.

FIG. 22. Nitrosylation of L-phenylalanine; [¹⁵ N]-NMR spectrum.

FIG. 23. UV spectrum for nitrosylation of tryptophan.

23a: 5 minute reaction time.

23b: 10 minute reaction time.

23c: 15 minute reaction time.

23d: 30 minute reaction time.

23e: 60 minute reaction time.

FIG. 24. [¹⁵ N]NMR for nitrosylated bovine serum albumin.

FIG. 25. UV spectrum for time-dependent NO loading of BSA.

25a: 1 minute reaction time.

25b: 5 minute reaction time.

25c: 30 minute reaction time.

FIG. 26. Nitrosylation of t-PA.

FIG. 27. Vasodilatory effects of NO-loaded BSA.

FIG. 28: S-nitrosylation of hemoglobin.

FIG. 29: UV spectrum of hemoglobin incubated withS-nitro-N-acetycysteine.

FIG. 30: Reaction of nitric oxide at the iron-binding site ofhemoglobin.

DESCRIPTION OF THE PREFERRED EMBODIMENTS Background

The invention is based on the discovery by the inventors thatnitrosylation of proteins and amino acids provides a means by whichprotein and amino acid function may be selectively regulated, modifiedor enhanced.

The term "nitrosylation" refers to the addition of NO to a thiol group(SH), oxygen, carbon or nitrogen by chemical means. The source of NO maybe endogenous NO or endothelium-derived relaxing factor, or othernitrosylating agents, such as nitroglycerin, nitrosothiols,nitrosothiols, nitrous acid or any other related compound.

The term "regulated" means effective control of the activity of aprotein or amino acid, in a selective manner so as to cause the proteinor amino acid to exert a desired physiological effect.

The term "modified" means to effectively alter the activity of a proteinor amino acid in a selective manner, so as to cause the protein or aminoacid to exert a desired physiological effect. The term "enhanced" meansto alter effectively the activity of a protein or amino acid in aselective manner, so as to cause an increase or improvement in theactivity of the protein or amino acid, or endow the protein or aminoacid with additional capabilities.

The term "activity" refers to any action exerted by the protein or aminoacid which results in a physiological effect.

The inventors have investigated the reaction of NO with protein thiolsand have demonstrated that a variety of proteins of biologicalsignificance and relative abundance can be S-nitrosylated.S-nitrosylation of proteins endows these molecules with potent andlong-lasting NO-like effects of vasodilation and platelet inhibition,mediated by guanylate cyclase activation, and also provides a means forachieving selective regulation of particular protein functions.

To develop the S-nitroso-protein compounds of the invention, certainthiol-containing proteins which are representative of various functionalclasses were nitrosylated. Such proteins include enzymes, such astissue-type plasminogen activator (t-PA) and cathepsin B; transportproteins, such as lipoproteins, hemoglobin, and serum albumin; andbiologically protective proteins, such as immunoglobulins.

The data demonstrate that 1) NO can react with thiol groups in proteinsto form S-nitrosothiols; 2) this reaction occurs under physiologicconditions; 3) these compounds are biologically active, exhibitingvasodilatory and anti-platelet properties that are independent of theirmethod of synthesis: 4) the long chemical half-lives ofS-nitroso-proteins vis-a-vis the half life of NO is reflected in theirdifferent relaxation kinetics: S-nitroso-proteins, through activation ofguanylate cycle, is fully consistent with that of othernitroso-compounds; although the possibility of other mechanisms by whichS--NO-proteins can produce biologic effects cannot be excluded, such asthe transfer of NO to another protein thiol, the function of which isthereby modulated. (Craven et al. J. Biol. Chem. 253:8433 (1978);Katsuki et al. J. Cyc. Nuc. Prot. Phos. Res. 3:23 (1977); Osborne etal., J. Clin. Invest. 83:465 (1989)).

Particular Embodiments

One embodiment of the invention relates to S-nitroso-enzyme compounds,derived from nitrosylation of enzymatic proteins.

A particular aspect of this embodiment relates to the compound,S-nitro-t-PA (S--NO-t-PA), derived from the nitrosylation of tissue-typeplasminogen activator (t-PA).

Acute occlusive events are precipitated by thrombogenic stimuli andalterations in flow dynamics within the vessel. Platelet activation,augmented local vasoconstriction, and recruitment of the coagulationsystem each plays a major role in the subsequent development of athrombus (Marder et al., New Eng. J. Med. 318:1512, 1520 (1988)). t-PAis one of the products secreted by blood vessel endothelium, whichspecifically counteracts these thromobogenic mechanisms. t-PA, a serineprotease, converts plasminogen to plasmin on fibrin and plateletthrombi, which in turn induces fibrinolysis and platelet disaggregation.Loscalzo et al., New Engl. J. Med. 319(14):925-931 (1989); Loscalzo etal., J. Clin. Invest. 79:1749-1755 (1987).

Attempts have been made to improve the thrombolytic efficacy andpharmacological properties of plasminogen activators, such as t-PA. Inlight of the role of platelets in clot formation and in reocclusivevascular events, one major focus has involved the use of ancillaryantiplatelet therapy. Some success has been achieved with aspirin(ISIS-2 Lancet 2:349.360 (1988)), and other benefits are reported forseveral newer antiplatelet compounds (Gold, H. K. New Engl. J. Med.323:1483-1485 (1990)). Attempts have also been made to improve thefunctional properties of the plasminogen activator itself throughsite-directed mutagenesis and synthesis of hybrid molecules andbiochemical conjugates (Runge et al., Circulation 79:217-224 (1989);Vaughan et al., Trends Cardiovasc. Med. Jan/Feb: 1050-1738 (1991)).

Motivated by the need for a plasminogen activity with improvedthrombolytic efficacy and anti-thrombogenic properties, the inventorsdiscovered that nitrosylation of t-PA creates a new molecule(S--NO-t-PA) which has improved thrombolytic capability, (e.g., theenzymatic activity of the enzyme is enhanced) as well as vasodilatoryand platelet inhibitory effect. The inventors demonstrated thatS-nitrosylation significantly enhances the bioactivity of t-PA, withoutimpairing the catalytic efficiency or other domain-specific functionalproperties of the enzyme.

In particular, S-nitrosylation of t-PA at the free cysteine, cys 83,confers upon the enzyme potent antiplatelet and vasodilatory properties,without adversely affecting its catalytic efficiency or the stimulationof this activity by fibrin(ogen). In addition, the S-nitrosothiol groupdoes not appear to alter the specific binding of t-PA to fibrin(ogen) orthe interaction of t-PA with its physiological serine proteaseinhibitor, PAI-1. The proteolytic activity, fibrin(ogen)- bindingproperties and regions for interaction with PAI-1 reside in severalfunctional domains of the molecule that are linearly separate from theprobable site of S-nitrosylation in the growth factor domain (cys 83).Thus, chemical modification of t-PA by NO does not markedly alterfunctional properties of t-PA residing in other domains. In addition,S-nitrosylation enhances-the catalytic efficiency of t-PA againstplasminogen, and increases its stimulation by fibrinogen.

NO is highly labile and undergoes rapid inactivation in the plasma andcellular milieu. This suggests that the reaction between NO and theprotein thiol provides a means of stabilizing NO in a form in which itsbioactivity is preserved. Specifically, S--NO-t-PA is a stable moleculeunder physiologic conditions and, much like NO, is capable ofvasodilation and platelet inhibition mediated by cyclic GMP. StabilizingNO in this uniquely bioactive form creates a molecule with intrinsicvasodilatory, antiplatelet, and fibrinolytic properties, which enable itto counteract each of the major thrombogenic mechanisms.

Another aspect of this embodiment relates to the administration of--NO-t-PA as a therapeutic agent to an animal for the treatment andprevention of thrombosis. Current thrombolytic strategies are based onthe understanding of the endogenous mechanisms by which the endotheliumprotects against thrombogenic tendencies. In particular, plateletinhibition and nitrovasodilation are frequently used concomitanttherapies with which to enhance reperfusion by plasminogen activators aswell as to prevent rethrombosis (Gold, H. K. New Engl. J. Med.323:1483-1485 (1990); (Marder et al., New Engl. J. Med. 318:1512-1520(1988)).

Administration of S--NO-t-PA to a patient in need thereof provides ameans for achieving "fibrin-selective" thrombolysis, whilesimultaneously attenuating the residual thrombogenicity resulting fromsimultaneous platelet activation and thrombin generation duringthrombolysis. Furthermore, by virtue of its fibrin binding properties,S--NO-t-PA provides targeted delivery of the antiplatelet effects of NOto the site of greatest platelet activation, the actual fibrin-plateletthrombus. S--NO-t-PA has therapeutic application in the treatment orprevention of conditions which result from, or contribute to,thrombogenesis, such as atherothrombosis, myocardial infarction,pulmonary embolism or stroke.

In summary, S--NO-t-PA possesses unique properties that facilitatedispersal of blood clots and prevent further thrombogenesis. Thediscovery of this unique molecule provides new insight into theendogenous mechanism(s) by which the endothelium maintains vesselpatency and often a novel, and beneficial pharmacologic approach to thedissolution of thrombi.

Another aspect of this embodiment relates to the compounds derived fromthe nitrosylation of other thrombolytic agents, such as streptokinase,urokinase, or a complex containing one or more thrombolytic agents, suchas streptokinase, urokinase, or t-PA. These compounds may also beadministered to an animal, in the same manner as S--NO-t-PA for thetreatment and prevention of thrombosis.

An additional aspect of this embodiment relates to compounds derivedfrom the nitrosylation of other enzymes. One particular compound isS--NO-cathepsin, derived from the nitrosylation of cathepsin B, alysosomal cysteine protease. The invention have demonstrated thatS--NO-cathepsin exerts a vasodilatory and platelet inhibitory effect.Thus, this compound may be administered as a therapeutic agent to ananimal, to promote vasodilation and platelet inhibition, and to treat orprevent cardiovascular disorders.

Another embodiment of the invention relates to S-nitroso-lipoproteincompounds derived from the nitrosylation of lipoproteins. Suchlipoproteins include chylomicrons, chylomicron remnant particles, verylow-density lipoprotein (VDL), low-density lipoprotein (LDL),intermediate-density lipoprotein (IDL), and high density lipoprotein(HDL) and lipoprotein (a). The inventors have demonstrated thatS-nitroso-lipoproteins exert vasodilatory and platelet inhibitoryeffect. Thus, these compounds may be administered as a therapeuticagent, to an animal, to promote vasodilation and platelet inhibition,and to treat or prevent cardiovascular disorders.

An additional embodiment of the invention involves the in vivonitrosylation of lipoproteins as a means for regulating cellular uptakeof lipoproteins. Consequently, nitrosylation provides a means forregulating lipid uptake, and treating or preventing disorders associatedwith hyperlipidemias, such as atherosclerosis.

Another embodiment of the invention relates to theS-nitroso-immunoglobulin compounds derived from the nitrosylation ofimmunoglobulins. Such immunoglobulins may include IgG, IgM, IgA, IgD, orIgE. The inventors have demonstrated that these compounds exertvasodilatory and platelet inhibitory effect. Thus, these compounds maybe administered as therapeutic agents, to an animal, to promotevasodilation and platelet inhibition, and to treat or preventcardiovascular disorders. The half lives of these compounds, in theorder of one day, produce unique, long-lasting vasodilatory effectswhich are notably different from those of low molecular weightnitroso-compounds.

An additional embodiment of the invention is the compoundS-nitroso-hemoglobin, derived from the nitrosylation of hemoglobin. Thiscompound may be used as therapeutic agent to promote vasodilation andplatelet inhibition, and to treat or prevent cardiovascular disorders.

As demonstrated by the inventors, S-nitrosylation of hemoglobinincreases its oxygen-binding capacity. Hemoglobin is a globular protein,which binds reversibly to blood oxygen through passive diffusion fromentry of air into the lungs. Hemoglobin-oxygen binding greatly increasesthe capacity of the blood to transport oxygen to bodily tissues; thus,the binding affinity between hemoglobin and oxygen is a critical factorin determining the level of oxygen transport to the tissues. The thiolgroup on the hemoglobin molecule regulates the affinity of hemoglobinfor oxygen. The inventors have demonstrated that some S-nitrosothiols,such as S-nitroso-proteins do not react with the iron-binding site ofhemoglobin, as does NO•, but instead, bind to the thiol group. Thus,methemoglobin formation is prevented and hemoglobin-oxygen binding isunimpaired.

Furthermore, the inventors have also demonstrated that S-nitrosylationof hemoglobin not only prevents impairment of binding, but actuallyincreases hemoglobin-oxygen binding. Therefore, another embodiment ofthe invention involves the administration of S--NO-hemoglobin or the invivo nitrosylation of hemoglobin, to increase the oxygen-carryingcapacity of the blood, and oxygen transport to bodily tissues. As aresult, these compounds may be useful in the treatment of disorderswhich are associated with insufficient oxygen transport, or in clinicalsituations in which increased oxygen transport is needed. Examples ofsuch clinical situations include, but are not limited to, hypoxicdisorders resulting from pneumothorax, airway obstruction, paralysis orweakness of the respiratory muscles, inhibition of respiratory centersby drug or other agents, or other instances of decreased pulmonaryventilation. Additional clinical indications include impaired alveolargas diffusion such as occurs in interstitial fibrosis, bronchioleconstriction, pulmonary edema, pneumonia, hemorrhage, drowning, anemias,arteriovenous shunts, and carbon monoxide poisoning.

In addition, S--NO-hemoglobin may also be used to modulate the deliveryof carbon monoxide or nitric oxide (bound to hemoglobin) to bodilytissues.

In addition, any thiol-containing heme proteins may be nitrosylated andused to enhance the oxygen-carrying capacity of the blood.

An additional embodiment of the invention is the compoundS-nitroso-myoglobin, derived from the nitrosylation of myoglobin, aprotein which also transports oxygen. This compound may be used as atherapeutic agent to promote vasodilation and platelet inhibition, andto treat or prevent cardiovascular disorders.

Another embodiment of the invention relates to a method for usingS-nitroso-proteins as a means for providing targeted delivery of NO. Theterm "targeted delivery" means that NO is purposefully transported anddelivered to a specific and intended bodily site. In the same manner asS--NO-t-PA, S--NO-immunoglobulin can be modified, by cationicmodification of the heavy chain, to provide targeted delivery of NO tothe basement membrane of the glomerulus in the kidney. Successfuldelivery of four NO molecules per immunoglobulin have been directed tothe kidney basement membrane in this matter. Targeted delivery of NOprovides a means for achieving site-specific smooth muscle relaxation,or other NO-mediated effects. In addition, delivery may be for thepurpose of nitrosylation of various molecules present in the body. Forexample, S-nitroso-proteins would deliver NO, and thus nitrosylatehemoglobin or myoglobin in order to increase oxygen binding.

A significant advantage of S-nitroso-proteins is that they deliver NO inits most biologically relevant, and non-toxic form. This is critical,because the pharmacological efficacy of NO depends upon the form inwhich it is delivered. This is particularly true in airways, where highlevels of O₂ and O₂ reactive species predispose to rapid inactivation ofthe NO moiety. As demonstrated by the inventors, S-nitroso-proteinsdeliver NO as the charged species, nitrosonium (NO⁺) or nitroxyl (NO⁻),and not the uncharged NO radical (NO•). This is important because thecharged species behave in a very different manner from NO• with respectto chemical reactivity.

In contrast to NO•, nitrosonium and nitroxyl do not react with O₂ or O₂species, and are also resistant to decomposition in the presence ofredox metals. Consequently, administration of NO equivalents does notresult in the generation of toxic by-products or the elimination of theactive NO moiety. By delivering nitrosonium or nitroxyl,S-nitroso-proteins provide a means for achieving the smooth musclerelaxant and anti-platelet effects of NO, and at the same time,alleviate significant adverse effects previously associated with NOtherapy.

Another embodiment of the invention relates to the administration ofS-nitroso-albumin as a therapeutic agent to promote platelet inhibition,or to cause relaxation of airway smooth muscle. The inventors havedemonstrated that S-nitroso-BSA exerts a platelet inhibitory effect, andalso promotes long-acting vasodilatory effect, which can bedistinguished from that of NO or the low molecular weight thiols.

The inventors have also demonstrated that S-nitroso-BSA relaxes humanairway smooth muscle. As discussed above, by delivering NO in the formof charged NO equivalents, such as nitrosonium, S-nitroso-proteins causeairway relaxation, and also eliminate the adverse effects which occurwith administration of other NO species. Thus, S-nitroso-albumin may beadministered for the treatment or prevention of respiratory disordersincluding all subsets of obstructive lung disease, such as emphysema,asthma, bronchitis, fibrosis, excessive mucous secretion and lungdisorders resulting from post surgical complications. In addition thesecompounds may be used as antioxidants, and thus, in the treatment ofdiseases such as acute respiratory distress syndrome (ARDS).

Another embodiment of the invention relates to a method fornitrosylation of those proteins which lack free thiols. The methodinvolves thiolating the protein by chemical means, such as homocysteinethiolactone (Kendall, BBA 257:83 (1972)), followed by nitrosylation inthe same manner as the compounds discussed above. Recombinant DNAmethods may also be used to add or substitute cysteine residues on aprotein.

Another embodiment of the invention relates to a method fornitrosylation of those proteins in which the thiol is blocked by amethyl group. The method involves selective de-methylation of theprotein by chemical means, such as reacting with methyl transferase,followed by nitrosylation in the same manner as the compounds discussedabove.

Another embodiment of the invention involves the use ofS-nitroso-protein compounds to relax non-vascular smooth muscle. Typesof smooth muscle include, but are not limited to, bronchial, tracheal,uterine, fallopian tube, bladder, urethral, urethral, corpus cavernosal,esophageal, duodenal, ileum, colon, Sphincter of Oddi, pancreatic, orcommon bile duct.

An additional embodiment of the invention involves the in vivonitrosylation of protein thiols, by administration of a nitrosylatingagent as a pharmaceutical composition. In vivo nitrosylation provides ameans for achieving any of the physiological effects discussed above, orfor regulation of additional protein functions.

In addition to thiol groups, proteins and amino acids possess othersites which can be nitrosylated. For example, such sites may include,but are not limited to, oxygen, nitrogen, and carbon. Thus, anadditional embodiment of the invention relates to the nitrosylation ofadditional sites, such as oxygen, nitrogen, and carbon which are presenton proteins and amino acids, as a means for achieving any of thephysiological effects discussed above, or for regulation of additionalprotein or amino acid functions. The inventors have shown that aromaticamino acids, such as tyrosine, phenylalanine and tryptophan can benitrosylated at the hydroxyl, and amino groups, as well as on thearomatic ring, upon exposure to nitrosylating agents such as NaNO₂,NOCl, N₂ O₃, N₂ O₄ and NO⁺. Other amino acids, such as serine andthreonine may also be nitrosylated in the same manner.

The ability to bind NO to a variety of different sites on an amino acidor protein provides a greater concentration of NO, and thus may enhanceregulation of protein function, as well as other NO-mediated effectssuch as smooth muscle relaxation and platelet inhibition. Thus, anotherembodiment of the invention relates to the use of amino acids andproteins which contain numerous NO molecules, to regulate protein oramino acid function and to effect smooth muscle relaxation and plateletinhibition. Additional therapeutic uses of these compounds include thetreatment or prevention of such disorders as heart failure, myocardialinfarction, shock, renal failure, hepatorenal syndrome, post-coronarybypass, gastrointestinal disease, vasospasm of any organ bed, stroke orother neurological disease, and cancer.

Another embodiment of the invention relates to a method for using thesenitrosylated proteins and amino acids as a means for providing targeteddelivery of NO to specific and intended bodily sites. These compoundshave the capacity to deliver charged NO equivalents. For example, alkylnitrites having the formula X--CONO and containing a beta-electionwithdrawing group would be able to deliver these charged NO equivalents.

The hydroxyl group of tyrosine also plays a central role in a variety ofcell regulatory functions. For example, phosphorylation of tyrosine is acritical cell regulatory event. In addition, serine residues alsoprovide phosphorylation sites. Thus, a particular aspect of thisembodiment relates to the nitrosylation of amino acids such as tyrosineand serine, to regulate cellular process such as, but not limited to,phosphorylation.

Another embodiment of the invention relates to the use ofO-nitrosylation of tyrosine residues on bovine serum albumin as a methodfor achieving smooth muscle relaxation and platelet inhibition.

Another embodiment of the invention relates to the nitrosylation of t-PAat additional sites, such as oxygen. For example, O-nitrosylation oft-PA, in addition to conferring vasodilatory and platelet inhibitoryproperties, alters the pharmokinetics of t-PA in such a way as to makeit unavailable as a substrate for its natural inhibitor, PA-I.

Another embodiment of the invention relates to the administration of apharmaceutical composition comprised of any S-nitroso-protein, toinhibit platelet function, cause vasodilation, relax smooth muscle,deliver nitric oxide to specific targeted bodily sites, or for thetreatment or prevention of cardiovascular or respiratory disorders.

An additional application of the present invention relates to thenitrosylation of additional compounds such as peptides,neutrotransmitters, pharmacologic agents and other chemical compounds,as a therapeutic modality. For example, nitrosylation of dopamine, aneurotransmitters improves the cardiac profile of the drug, by enhancingafterload reduction and scavenging free radicals, while simultaneouslyinhibiting platelets and preserving renal blood flow. Nitrosylation ofepinephrine and related sympathomimetic drugs alters the half-life ofthe drug and affects its β-agonist selectivity.

The nitrosylated proteins and amino acids of the present invention, orthe nitrosylating agents may be administered by any means that effectthrombolysis, vasodilation, platelet inhibition, relaxation ofnon-vascular smooth muscle, other modification of protein functions ortreatment or prevention of cardiovascular disorders, or any otherdisorder resulting from the particular activity of a protein or aminoacid. For example, administration may be by intravenous, intraarterial,intramuscular, subcutaneous, intraperitoneal, rectal, oral, transdermalor buccal routes.

According to the present invention, a "therapeutically effective amount"of therapeutic composition is one which is sufficient to achieve adesired biological effect. Generally, the dosage needed to provide aneffective amount of the composition, in which can be adjusted by one ofordinary skill in the art, will vary, depending on the age, health,condition, sex, weight, and extent of disease, of the recipient. Inaddition, the dosage may also depend upon the frequency of treatment,and the nature of the effect desired.

Compositions within the scope of this invention include all compositionswherein the S-nitroso-protein or the nitrosylating agent is contained inan mount effective to achieve its intended purpose. While individualsneeds vary, determination of optimal ranges of effective mounts of eachcomponent is within the skill of the art. Typical dosage forms contain 1to 100 mmol/kg of the S-nitroso-protein. The dosage range for thenitrosylating agent would depend upon the particular agent utilized, andwould be able to be determined by one of skill in the ant.

In addition to the pharmacologically active compounds, the newpharmaceutical preparations may contain suitable pharmaceuticallyacceptable carriers comprising excipients and auxiliaries whichfacilitate processing of the active compounds into preparations whichcan be used pharmaceutically. Preferably, the preparations, particularlythose preparations which can be administered orally and which can beused for the preferred type of administration, such as tablets, dragees,and capsules, and also preparations which can be administered rectally,such as suppositories, as well as suitable solutions for administrationby injection or orally, contain preferably, about 0.01 to 5 percent,preferably from about 0.1 to 0.5 percent of active compound(s), togetherwith the excipient.

The pharmaceutical preparations of the present invention aremanufactured in a manner which is itself known, for example, by means ofconventional mixing, granulating, dragee-making, dissolving, orlyophilizing processes. Thus, pharmaceutical preparations for oral usecan be obtained by combining the active compounds with solid excipients,optionally grinding the resulting mixture and processing the mixture ofgranules, after adding suitable auxiliaries, if desired or necessary, toobtain tables or dragee cores.

Suitable excipients are, in particular, fillers such as sugars, forexample lactose or sucrose, mannitol or sorbitol, cellulose preparationsand/or calcium phosphates, for example tricalcium phosphate or calciumhydrogen phosphate, as well as binders such as starch, paste, using, forexample, maize starch, wheat starch, rice starch, potato starch,gelatin, tragacanth, methyl cellulose, hydroxypropylmethylcellulose,sodium carboxymethylcellulose, and/or polyvinyl pyrrolidone. If desired,disintegrating agents may be added such as the above-mentioned starchesand also carboxymethylstarch, cross-linked polyvinyl pyrrolidone, agar,or algenie acid or a salt thereof, such as sodium alginate. Auxiliariesare, above all, flow-regulating agents and lubricants, for example,silica, talc, stearic acid or salts thereof, such as magnesium stearateor calcium stearate, and/or polyethylene glycol. Dragee cores areprovided with suitable coatings which, if desired, are resistant togastric juices. For this purpose, concentrated sugar solutions may beused, which may optionally contain gum arabic, talc, polyvinylpyrrolidone, polyethylene glycol and/or titanium dioxide, lacquersolutions and suitable organic solvents or solvent mixtures. In order toproduce coatings resistant to gastric juices, solutions of suitablecellulose preparations such as acetylcellulose phthalate orhydroxypropymethyl-cellulose phthalate, are used. Dye stuffs or pigmentsmay be added to the tablets or dragee coatings, for example, foridentification or in order to characterize combinations of activecompound doses.

Other pharmaceutical preparations which can be used orally includepush-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a plasticizer such as glycerol or sorbitol. The push-fitcapsules can contain the active compounds in the form of granules whichmay be mixed with fillers such as lactose, binders such as starches,and/or lubricants such as lactose, binders such as starches, and/orlubricants such as talc or magnesium stearate and, optionally,stabilizers. In soft capsules, the active compounds are preferablydissolved or suspended in suitable liquids, such as fatty oils, orliquid paraffin. In addition, stabilizers may be added.

Possible pharmaceutical preparations which can be used rectally include,for example, suppositories, which consist of a combination of the activecompounds with a suppository base. Suitable suppository bases are, forexample, natural or synthetic triglycerides, or paraffin hydrocarbons.In addition, it is also possible to use gelatin rectal capsules whichconsist of a combination of the active compounds with a base. Possiblebase materials include, for example, liquid triglycerides, polyethyleneglycols, or paraffin hydrocarbons.

Suitable formulations for parenteral administration include aqueoussolutions of the active compounds in water-soluble form, for example,water-soluble salts. In addition, suspensions of the active compounds asappropriate oily injection suspensions may be administered. Suitablelipophilic solvents or vehicles include fatty oils, for example, sesameoil, or synthetic fatty acid esters, for example, ethyl oleate ortriglycerides. Aqueous injection suspensions may contain substanceswhich increase the viscosity of the suspension include, for example,sodium carboxymethyl cellulose, sorbitol, and/or dextran. Optionally,the suspension may also remain stabilizers.

Having now generally described the invention, the same will be morereadily understood through reference to the following examples which areprovided by way of illustration, and are not intended to be limiting ofthe present invention.

EXAMPLES Example 1: Synthesis of S-Nitroso-t-PA A. nitrosylation of t-PA

1. Materials

t-PA was kindly provided by Genentech, Inc. San Francisco, CA.Reactivated purified plasminogen activator inhibitor-1 (PAI-1) and apanel of six murine anti-t-PA monoclonal antibodies were kindly providedby Dr. Douglas E. Vaughan. Horse-Radish Peroxidase linked-sheepantimurine antibodies were purchased from Amersham Corp., Arlington, II.Sodium nitrite was purchased from Fisher Scientific, Fairlawn, NJ.H-D-isoleucyl-L-prolyl-L-arginyl-p-nilxoanflide (S2288) andH-D-valyl-L-leucyl-L-lysyl-p-nitroanilide (S2251) were purchased fromKabi Viman, Stockholm, Sweden. Human fibrinogen purified of plasminogenand von Willebrand factor, was obtained from Enzyme ResearchLaboratories, South Bend, IN. Epinephrine, ADP and iodoacemmide werepurchased from Sigma Chemical Co., St. Louis, MO. Bovine thrombin wasobtained from ICN, ImmunoBiologicals (Lisle, IL). Radioimmunoassay kitsfor the determination of cGMP were purchased from New England Nuclear,Boston, MA.

2. Plasminogen Preparation

Glu-plasminogen was purified from fresh frozen plasma thawed at 37° C.using a modification of the method of Deutsch and Mertz (Deutsch et al.,Science 170:1095-1096 (1970), herein incorporated by reference). Plasmawas passed over a lysine--Sepharose column and the column washed with0.3 M sodium phosphate, pH 7.4, 3 mM EDTA. Plasminogen was eluted fromthe column with 0.2M epsilon-aminocaproic acid, 3 mM EDTA, pH 7.4.Contaminant plasmin was removed by passing the eluted column overbenzamidine sepharose 2B. The plasminogen obtained was subsequentlydialyzed before use against 10 mM sodium phosphate, pH 7.4, 0.15M NaCl.

3. Thiol Derivatization

The free thiol of t-PA was carboxyamidated by exposure of the enzyme toa 10-fold excess of iodoacetamide in the dark for one hour at 37° C. in10 mM Tris, pH 7.4, 0.15M NaCl (TBS). t-PA was then dialyzed extensivelyagainst 10 mM HCl in order to remove excess iodoacetamide.

4. Microcarrier Endothelial Cell Culture

Endothelial cells were isolated from bovine aorta by establishedtechniques (Schwartz, S. M. In Vitro 14:966-980 (1978), hereinincorporated by reference) and cultured on a microcarrier system ofnegatively charged spherical plastic beads (Biosilon), according to themethod of Davies and colleagues (Davies et al., J. Cell Biol.101:871-879 (1985), herein incorporated by reference).

5. Nitrosylation

t-PA was first dialyzed against a large excess of 10 mM HCl for 24 hoursto remove excess L-arginine used to solubilize the protein. t-PA wasthen exposed to NO_(x) generated from equimolar NaNO₂ in 0.5N HCl(acidified NaNO₂) or in control experiments, to 0.5N HCl alone, for 30minutes at 37° C. Solutions were titrated to pH 7.4 with equal volumesof 1.0 N NaOH and Tris Buffered Saline (TBS), pH 7.4, 0.05M L-arginine.Dilutions were then made as necessary in TBS.

For comparative purposes, and to illustrate the potential biologicalrelevance of S--NO-t-PA, this compound was synthesized with authenticEDRF in selected experiments. In this method, t-PA was incubated withbovine aortic endothelial cells stimulated by exposure to high shearforces to secrete EDRF, as we have previously described (Stamler et al.,Cir. Res. 65:789 (1989), herein incorporated by reference). Owing to thestability of the S--NO bond in S--NO-t-PA under physiologic conditions(t.sub. 1/2 >24 hours in TBS, pH 7.4, 20° C.), samples were stored at pH7.4 on ice throughout the course of the experiments.

S--NO-t-PA has also been synthesized by exposure of t-PA to NO gasbubbled into buffered (TBS) solution of enzyme. This further illustratesthe potential for s-nitrosylation, by exposure of proteins to a varietyof oxides of nitrogen including NOCl, N₂ O₃, N₂ O₄ and othernitroso-equivalents.

B. Confirmation of S--NO bond

1. Methods

The formation of and stability of the S--NO bond were was confirmed byseveral published analytical methods.

In the first, NO displaced from S-nitrosothiol groups with Hg²⁺ wasassayed by diazotization of sulfanilamide and subsequent coupling withthe chromophore N-(1-naphthyl-)ethylenediamine (Saville, B. Analyst83:670-672 (1958), herein incorporated by reference). In the second, thecharacteristic absorption spectrum of S-nitrosothiols in the range of320 nm-360 nm was detected (Stamler et al., Proc. Natl. Acad. Sci. USAin press (1991); Oac et al., Org. Prep. Proc. Int. 15(3):165-169(1983)).

In the third, [¹⁵ N] NMR was used. Measurements of RS--NOs were madeaccording to the method of Bonner and colleagues (Bonnett et al., ICSPerkins Trans. 1:2261-2264 (1975), herein incorporated by reference).[¹⁵ N]NMR spectra were recorded with a Brucker 500 MHZ spectrometer,Billerica, MA. Deuterium lock was effected with [D]₂ O and the spectrareferenced to an [¹⁵ N] natural abundance spectrum of a saturatedsolution of NaNO₂ at 587 ppm. Spectra were recorded at 50.68 MHZ and thenine transients of 16k data points collected with a 30° pulse width anda 10-second relaxation delay. Data were multiplied by a 2-Hz exponentialline broadening factor before Fourier transformation.

Confirmation of the above chemical evidence for protein S-nitrosothiolsynthesis was obtained by UV, NMR and IR spectroscopy. Previouscharacterization of S-nitrosothiols, revealed that they passes UVabsorption maxim at 320-360 nm, chemical shifts of approximately 750 ppmrelative to nitrite (Bonnett et al., JCS Perkins Trans. 1:2261-2264(1975)), and IR stretches at approximately 1160 cm⁻¹ and 1170 cm⁻¹ cm.(Loscalzo et al., JPET 249:726-729 (1989)).

2. Results

In accordance with these observations, S--NO-t-PA exhibited anabsorption maximum at 322 nm (FIG. 1a), and a chemical shift at 751 ppm(relative to nitrite) (FIG. 1b); elimination of the chemical shift wasachieved by sample treatment with excess HgCl₂. In addition, thepresence of two absorption bands at 1153 cm⁻¹ and 1167 cm⁻¹, is entirelyconsistent with the formation of an S-nitrosothiol bond (Myers et al.,Nature 345:161-163 (1990); Oac et al., Org. Prep. Proc. Int.15(3)165-169 (1983); Bonnett et al., JCS Perkins Trans. 1:2261-2264(1975). The quantification of NO (Protein-NO+free NO_(x)) in the Savillereaction, and the NMR results demonstrating a single chemical shift,reveal that all NO bound to the protein exists in the form of anS-nitrosothiol.

FIG. 2 illustrates the time-dependent formation of S--NO-t-PA. Aliquotsof the solution containing NaNO₂ were removed sequentially fordetermination of --S--NO bond formation (Schwartz, S. M. In Vitro14:966-980 (1978)). Results are expressed as mean ±S.D. (n=3). By 30minutes of exposure to acidified NaNO₂, S-nitrosylation is essentiallycomplete; the stoichiometry of --S--NO/t-PA (mol/mol) is 0.0±0.1 (n=3)at the completion of the reaction as determined by the method of Saville(Saville, B. Analyst 83:670-672 (1958)). Carboxyamidation of t-PA's freethiol with iodoacetamide completely prevents S-nitrosothiol formation asdetermined by this chemical method (Saville, B. Analyst 83:670472(1958)).

FIG. 2 also illustrates the effect of acid treatment on the amidolyticactivity of t-PA. At different intervals, aliquots of the enzyme exposedto 0.5 N HCl alone were neutralized, and amidolytic activity was assayedusing the chromogenic substrate S2288. Results are expressed as mean±S.D. (n=3), relative to t-PA not treated with 0.5N HCl. At 30 minutes,the duration of exposure subsequently used for S-nitrosothiol synthesis,the enzymatic activity of t-PA is largely preserved. Quantification ofS--NO-t-PA synthesis with authentic EDRF was similarly determined by themethod of Saville (Saville, B. Analyst 83:670472 (1958)).

Example 2: Synthesis of S-Nitroso-BSA A. Nitrosylation

In the first method, nitrosylation of BSA was accomplished by incubatingBSA (200 mg/ml with NO generated from equimolar NaNo₂ in 0.5N HCl(acidified NaNO₂) for thirty minutes at room temperature. Solutions weretitrated to pH 7.4 with equal volumes of 1.0N NaOH and Tris BufferedSaline (TBS), pH 7.4, 0.05M L-arginine. Dilutions were then made asnecessary in TBS.

In the second method, nitrosylation was achieved in helium-deoxygenatedsolutions of 0.1M sodium phosphate (pH 7.4) by exposing the proteinsolution in dialysis tubing to authentic NO gas bubbled into thedialysate for fifteen minutes. The proteins were then dialyzed against alarge excess of 0.01M phosphate buffer at pH 7.4 to remove excess oxidesof nitrogen.

In the third method, proteins were incubated with bovine aorticendothelial cells stimulated by exposure to high shear forces to secreteEDRF, as in Example 1(A). As a corollary of this method, proteins werealso incubated directly with NO synthase purified from bovine cerebellum(Bredt et al., Proc. Natl. Acad. Sci. USA 87:682 (1990), hereinincorporated by reference) in the presence of the substrate L-arginineand cofactors required for enzyme activity (Ca⁺⁺, calmodulin, andNADPH).

B. Confirmation of S-nitroso-protein formation

The formation and stability of the S-nitroso-protein was confirmed byseveral published analytical methods. NO displaced from S-nitrosothiolgroups with Hg²⁺ was assayed by diazotization of sulfanilamide andsubsequent coupling with the chromophore N-(1-naphthyl-ethylenediamine(Mellion et al., Mol. Pharmacol. 23:653 (1983); Saville, B. Analyst83:670 (1958)). The stoichiometries of S--NO-BSA determined by thesechemical methods is shown in Table 1.

Confirmatory evidence for S-nitrosothiol bond formation in proteins wasobtained by spectrophotometry; S-nitrosothiols possess dual absorptionmaxima at 320-360 nm and at approximately 550 nm (Oae et al., OrganicPrep. and Proc. Int. 15:165 (1983); Ignarro et al., J. Pharmacol. Exp.Ther. 218:739 (1981); Mellion et al., Mol. Pharmacol. 23:653 (1983);Loscalzo, J., Clin. Invest. 76:966 (1985)).

As one additional, more specific measure of protein S-nitrosylation, [¹⁵N]-NMR spectroscopy was used. BSA was S-nitrosylated with Na[¹⁵ N]O₂ andthe [¹⁵ N]-NMR spectrum of the resulting species recorded in FIG. 3.FIG. 3 demonstrates the [¹⁵ N]-NMR spectrum of [¹⁵ N]-labeledS-nitroso-BSA. The chemical shift for S-nitroso-BSA was 703.97, whichfalls into the same range as other S-nitrosothiols (e.g.,S-nitroso-L-cysteine) prepared under similar conditions (Bonnett et al.,J. Chem. Soc. Perkins Trans. 1:2261 (1975)). The spectrum was recordedat 50.68 MHZ and the nine transients of 16K data points were collectedwith a 30° pulse width and a 2.5-sec relaxation delay. Data weremultiplied by a 2-Hz exponential line broadening factor before Fouriertransformation. The region of 590 to 810 ppm is displayed.

Example 3: Synthesis of S-Nitroso-Cathepsin B

Nitrosylation of cathepsin, and determination of S-nitrosothiolformation, was accomplished according to the methods described inExample 2. The stoichiometry of S-nitrosothiol/protein molecules forcathepsin is shown in Table 1.

Example 4: Synthesis of S-Nitroso-Lipoprotein

Synthesis was accomplished by nitrosylating purifiedlow-density-lipoprotein (LDL) according to the methods described inExample 2. Confirmation of S-nitroso-protein formation was verifiedaccording to the methods of Example 2. The stoichiometry ofS-nitrosothiol/protein molecules for LDL is shown in Table 1.

Example 5: Synthesis of S-Nitroso-Immunoglobulin

Synthesis was accomplished by nitrosylating purified gamma globulin(Sigma) according to the methods described in Example 2. Confirmation ofS-nitroso-protein formation was verified according to the methods ofExample 2, The stoichiometry of S-nitrosothiol/protein molecules forimmunoglobulin is shown in Table 1.

                  TABLE I                                                         ______________________________________                                        S-NITROSO-PROTEIN SYNTHESIS                                                                   --S--NO/protein (mol/mol)                                     ______________________________________                                        Bovine Serum Albumin                                                                            0.85 ± 0.04                                              t-PA              0.88 ± 0.06                                              Cathepsin B       0.90 ± 0.02                                              Human plasma      0.87 ± 0.02                                              Immunoglobulin    0.35 ± 0.01                                              Lipoprotein (LDL) 1.80                                                        ______________________________________                                          Legend-                                                                      The stoichiometries for the indiviual --S--NO/protein molar ratios are        given in the table and represent the mean ± SEM of 3 to 6                  determinations.                                                          

Example 6: Demonstration of Thrombolytic, Anti-Platelet And VasodilatoryEffect of S--NO-t-PA A. Thrombolysis

1. Fibrinogen Binding

The binding of t-PA and S--NO-t-PA to fibrinogen was measured usingpolystyrene microliter wells (flat-bottom, high binding 96-well EIAplates, cat. #3590, Costar, Cambridge, MA). Wells were coated withfibrinogen (0.08 ug/ul) and the remaining binding sites with 2% bovineserum albumin. Quantification of t-PA binding was determined using aHorse-Radish Peroxidase linked sheep antimurine antibody in acolorimetric assay in the presence of O-phenylenediamine, 0.014% H₂ O₂.Color change was measured spectrophotometrically with a Dynatech MR500Card Reader (Dynatech, Chantilly, VA) at 490 nm.

Binding of t-PA is reversible and specific, and saturates at 1500-3000nM; at saturation, 18 ng of t-PA are bound per well (0105 moles t-PA permole of fibrinogen) with an estimated K_(D) in the range of 15-650 nM.Binding of t-PA and S--NO-t-PA was quantified by ELISA over theconcentration range of 150-1500 nM using a mixture containing six murinemonoclonal anti-t-PA antibodies.

a. Comparison of t-PA and S--NO-t-PA

The binding of t-PA to fibrin(ogen) accounts for the relative"fibrin-specificity" of the enzyme as compared to certain otherplasminogen activators (Loscalzo et al., New Engl. J. Med.319(14):925-931 (1989); Vaughan et al., Trends Cardiovasc. Med. Jan/Feb:1050-1738 (1991)). The effect of S-nitrosylation on this functionalproperty of the enzyme was therefore assessed. The binding isotherms fort-PA and its S-nitrosylated derivatives were not significantly differentfrom each other by two-way ANOVA. Therefore, these data were subjectedto a single best-curve-fit binding isotherm (FIG. 4). From a Scatchardanalysis, the estimated apparent D_(D) of S--NO-t-PA for surface-boundfibrinogen is 450 nm, which falls well within the reported range fort-PA (Ranby, M. Blochim. Biophysica Acta 704:461-469 (1982)).

2. Measurement of Enzymatic Activity

The amidolytic activities of t-PA and its S-nitrosylated derivative weremeasured using the relatively specific chromogenic substrate, S2288.Substrate hydrolysis was measured spectrophotometrically at 405 mm witha Gilford Response UV/Vis Spectrophotometer (CIBA-Corning, Oberlin, OH).Activity was measured at 25° C. in TBS using substrate concentrationsvarying from 0.1-2.0 mM and t-PA at a concentration of 100 nM. Kineticparameters were determined from initial rates by double reciprocal plotanalysis. The assessment of inhibition of t-PA and S--NO-t-PA enzymaticactivity by PAI-1 was made at an enzyme concentration of 10 nM and amolar ratio of t-PA to active PAI-1 of 1.0. The degree of inhibition wasdetermined relative to the initial rates in the absence of theinhibitor.

In the coupled enzyme assay, t-PA and S--NO-t-PA activities were assayedusing the native substrate S2251. In selected experiments, fibrinogenstimulation of enzymatic activity was assessed at a fibrinogenconcentrations of 1 mg./ml. Substrate hydrolysis was measuredspectrophotometrically with a Dynatech MR 5000 Card Reader (Dynatech,Chantilly, VA) in TBS, pH 7.4, at 25° C. Initial reaction velocity wasdetermined from the slope of the plot of absorbance (at 405 nm)/time vs.time (Ranby, M. Biochim. Biophysica Acta 704:461-469 (1982)) usingglu-plasminogen concentrations ranging from 0.1-10 μM at an S2251concentrations of 0.8 mM. Kinetic parameters were determined frominitial rates by double reciprocal plot analysis.

a. Comparison of t-PA and S--NO-t-PA

The amidolytic activity of t-PA and S--NO-t-PA were first comparedagainst the chromogenic substrate S2288. From a double reciprocal plotanalysis it is evident that the kinetic parameters (K_(m) and V_(max))and the catalytic efficiency (K_(cat) /K_(m)) of these molecules areessentially identical, as shown in FIG. 5a. The values of these kineticconstants are provided in Table 2.

The effect of S-nitrosylation on the ability of t-PA to activate itsphysiologic substrate, plasminogen, was assessed in the coupled enzymeassay in the presence and absence of fibrinogen. As seen in theLineweaver-Burke plot (FIG. 5b) and from the derived kinetic parameters(Table 2), S--NO-t-PA has a K_(m) for substrate similar to "wild type"t-PA. However, S--NO-t-PA has a slightly, but significantly, greaterV_(max) yielding a catalytic efficiency that is 23% greater than that ofnative t-PA.

3. Discussion

Both fibrin and fibrinogen increase the rate of activation ofplasminogen by t-PA. The enhanced enzymatic activity of t-PA is theresult of its ability to bind directly fibrin(ogen), which brings abouta confromational change either in t-PA or plasminogen that promotes theinteraction of t-PA with its substrate (Loscalzo et al., New Engl. J.Med. 319(14):925-931 (1989)).

The consequences of S-nitrosylation on these important functionalproperties of t-PA were therefore studied in a comparative analysis witht-PA in the coupled enzyme assay. The results, summarized in FIG. 6,indicate that S--NO-t-PA binds to fibrinogen; that as a result of thisbinding its enzymatic activity is enhanced; and that in the presence ofphysiologic (1 μM) plasminogen concentrations, the degree of stimulationis equivalent to that of "wild type" t-PA. At lower plasminogenconcentrations (0.1 μM), fibrinogen stimulation of S--NO-t-PA was3.5-fold greater than t-PA (1 μM) (p<0.05). Absolute rates ofplasminogen activation were again slightly greater for SO--NO-t-PA (v/dasupra).

t-PA is rapidly inhibited by its cognate plasma serpin, PA1-1 (Loscalzoet al., New Engl. J. Med. 319(14):925-931 (1989); Vaughan et al., TrendsCardiovasc. Med. Jan/Feb:1050-1738 (1991)). By serving as apseudo-substrate, PAI-1 reacts stoichiometrically with t-PA to form aninactive complex. PAI-1 was equally effective at inhibiting thehydrolytic activity of t-PA and S--NO-t-PA in the direct chromogenicassay with S2288 (n-3; P--NS). Thus, S-nitrosylation of t-PA does notappear to alter its interaction with PAI-1.

B. Platelet Inhibition

1. Preparation of Platelets

Venous blood, anticoagulated with 1-mM trisodium citrate, was obtainedfrom volunteers who had not consumed acetylsalicylic acid for at leastten days. Platelet-rich plasma (PRP) was prepared by centrifugation at150 g for ten minutes at 25° C. Platelet counts were determined with aCoulter counter (model ZM; Coulter Electronics, Hialeah, FL).

2. Platelet Gel-Filtration and Aggregation

Platelets were gel-filtered on a 4×10 cm column of Sepharose 2B inTyrode's Hepes buffer as described previously (Hawiger et al., Nature2831:195-198 (1980), herein incorporated by reference). Platelets weretypically suspended at a concentration of 1.5×10⁸ /ml and were usedwithin 30 minutes of preparation. Platelet aggregation was monitoredusing a standard nephelometric technique (Born, et al., J. Physiol.168:178-195 (1963), herein incorporated by reference), in which 0.3-mlaliquots of gel-filtered filtered platelets were incubated at 37° C. andstirred at 1000 rpm in a PAP-4 aggregometer (Biodata, Hatboro, PA).Gel-filtered platelets were preincubated with t-PA or S--NO-t-PA for upto 45 minutes and aggregations induced with 5 μM ADP or 0.025 U/mlthrombin.

Aggregations were quantified by measuring the maximal rate or extent oflight transmittance and expressed as a normalized value relative tocontrol aggregations.

3. Cyclic Nucleotide Assays

The antiplatelet actions of S-nitrosothiols are mediated by cyclic GMP.Measurements of cGMP were performed by radioimmunoassay. Gel-filteredplatelets were pre-incubated for 180 seconds with S--NO-t-PA (9 μM), andrelated controls. Reactions were terminated by the addition of 10%trichloracetic acid. Acetylation of samples with acetic anhydride wasused to increase the sensitivity of the assay.

S--NO-t-PA incubated with platelets for 180 seconds, induced an 85%increase in inwacellular cyclic GMP above basal levels in the presenceof t-PA (p<0.01). The elevation in intracellular platelet cGMP inducedby S--NO-t-PA was entirely prevented by intracellular of platelets withthe guanylate cyclase inhibitor methylene blue (10 μM for ten minutes(n=3) (FIG. 7).

4. Discussion

The effects of S--NO-t-PA were studied in a gel-filtered plateletpreparation. In these experiments, NO_(x) generated for NaNO₂ had nosignificant effect on the extent of platelet aggregation (tracing notshown). Mean results for inhibition by S--NO-t-PA are presented in Table4.

FIG. 8 illustrates platelet inhibition induced by S--NO-t-PA (333 nM)synthesized with EDRF. In these experiments, t-PA was exposed toendothelial cells stimulated to secrete EDRF for 15 minutes after whichthe formation for S--NO-t-PA was verified by method for Saville(Saville, B. Analyst 83:670-672 (1958)). S--NO-t-PA was thenpreincubated with platelets for ten minutes prior to induction ofaggregation with 5 μM ADP. In the absence of t-PA, effluent fromendothelial cells stimulated to secrete EDRF had no significant effecton platelet aggregation. S--NO-t-PA inhibited platelet aggregation to 5μM ADP in a dose-dependent manner, with 50±16% (mean ±S.D.) inhibitionin ram and extent of aggregation observed at 1.4 μM S--NO-t-PA (n=4;p<0.001 vs. control). Inhibition of platelet aggregation induced by ADP(5 μM) or thrombin (0.024 U/ml) was demonstrable at concentrations ofS--NO-t-PA in the pharmacologic range of 15-150 nM, as shown in theillustrative tracings of FIG. 8(a) and (b) and in Table 4. In furthersupport of the potential biological relevance for RS--NOs, and thecomparable bioactivity of S--NO-t-PA irrespective of its method ofsynthesis, inhibition of platelet aggregation by S--NO-t-PA (33 nM)synthesized with authentic EDRF is illustrated in FIG. 8(c).

C. Vasodilation

1. Preparation of Blood Vessels

New Zealand White female rabbits weighing 3-4 kg were anesthetized with30 mg/kg IV sodium pentobarbital. Descending thoracic aortae wereisolated and placed immediately in a cold physiologic salt solution(Kreb's) (mM): NaCl, 118: CKl, 4.7; CaCl₂, 2.5; MgSO₄, 1.2; KH₂ PO₄,1.2; NaHCO₃, 12.5; and D-glucose, 11.0. The vessels were cleaned ofadherent connective tissue, and the endothelium removed by gentlerubbing with a cotton tipped applicator inserted into the lumen, afterwhich the vessel was cut in 5 mm rings. The rings were mounted onstirrups and connected to transducers (model FT03C Grass Instruments,Quincy, MA) by which changes in isometric tension were records.

2. Bioassay

Samples were added to a standard bioassay in which vessel tings weresuspended in glass chambers containing seven ml of oxygenated Kreb'sbuffer, in a standard bioassay (Cook et al., Am. J. Physiol. 28:H804(1989), herein incorporated by reference). Sustained contractions, to 2gm tension, were induced with 1 μM epinephrine, after which the effectsof t-PA and S--NO-t-PA were tested. In certain experiments the guanylatecyclase inhibitor, methylene blue, was preincubated with vessel ringsfor 15 minutes prior to initiation of contractions.

3. Vascular Relaxations

As shown in the illustrative tracings of FIG. 9, S--NO-t-PA, atpharmacologic concentrations, induces relaxations that are unmatched byequimolar mounts of the reactant protein-thiol or NO alone. Furthermore,consistent with the mechanism of other nitro(so)-vasodilators,relaxations were attenuated by the guanylate cyclase inhibitor,methylene blue. Table 3 depicts the effect of S--NO-t-PA on vesselrelaxation for several such experiments.

                  TABLE 2                                                         ______________________________________                                        Kinetic Parameters of S2288 Hydrolysis                                        and GLU-Plasminogen (S2251)                                                   Activation By t-PA and S--NO-t-PA                                                     K.sub.m   k.sub.cat                                                                             k.sub.cat /K.sub.M                                          (μM)   (sec.sup.-1)                                                                          (sec.sup.-1 - μM.sup.-1                          ______________________________________                                        S2288                                                                         t-PA      280         0.52    0.0019                                          S--NO-t-PA                                                                              295         0.52    0.0019                                          S2251                                                                         t-PA      3.5         0.200   0.056                                           S--NO-t-PA                                                                              3.8         0.262   0.069                                           ______________________________________                                    

                  TABLE 3                                                         ______________________________________                                        VESSEL RELAXATION                                                                                  % Relaxation                                             ______________________________________                                        t-PA           (150 nM)    2.5 ± 4                                         NO             (150 nM)    1.0 ± 1.7                                       S--NO-t-PA     (150 nM)     20 ± 7*                                        ______________________________________                                         Mean results (± S.D.; n = 4) of vessel relaxation induced by S--NOt-PA     and the comparable relaxation induced by equivalent concentrations of NO      (generated from acidified NaNO.sub.2) a tPA.                                  *Relaxations to S--NOt-PA were significantly greater than those induced b     NaNO.sub.2 or tPA, as shown in this table for equal concentrations.      

                  TABLE 4                                                         ______________________________________                                        PLATELET INHIBITION                                                                  % Normalized Extent Aggregation                                                               Thrombin (0.024                                                     ADP (5 μM)                                                                           U/ml)                                                  ______________________________________                                        t-PA     (150 μM)                                                                             1.06 ± 0.24                                                                            0.90 ± 0.15                                 S--NO-t-PA                                                                             (150 μM)                                                                             0.77 ± 0.28†                                                                    0.73 ± 0.28*                                ______________________________________                                         Mean results (± S.D.; n = 13-17) of platelet inhibition mediated by        S--NOt-PA to both ADinduced platelet aggregation. NO generated from           NaNO.sub.2 (150 nM) had no significant effect of platelet inhibition in       these experiments (0.98 ± 0.11, n = 5).                                    *p < 0.025 compared with tPA;                                                 †p < 0.01 compared with tPA.                                      

Statistics

Determination of statistical significance was analyzed using a nonpairedt-test or two-way analysis of variance (ANOVA) followed by aNewman-Keul's comparison.

Example 7: Demonstration of Platelet Inhibitory and Vasodilatory Effectof S-Nitroso-BSA A. Platelet Inhibition

The effect of S-nitroso-BSA on platelet aggregation was studied, using agel-filtered platelet preparation, as previously described (Hawiger etal., Nature 2831:195 (1980)) and suspended at 150,000 platelets/ul inHEPES buffer, pH 7.325. S--NO-BSA was incubated with platelets for tenminutes at 37° C. in a PAP-4 aggregometer (BioData, Hatboro, PA), afterwhich aggregations were induced with 5 μM ADP. Aggregations werequantified by measuring the extent of change of light transmittance andexpressed as a normalized value relative to control aggregations.

In control experiments, neither NaNO₂ at concentrations up to 15 μM northe effluent from cells stimulated to secrete EDRF in the absence of BSAhad any significant effect on either vessel tone or plateletaggregation. All non-nitrosylated proteins studied had no significanteffect on platelet aggregation at any concentration tested.

Dose-dependent inhibition of ADP-induced platelet aggregation wasobserved over the range of 150 nM to 15 μM S-nitroso-protein. Anitrosylated protein plasma fraction was even more potent, manifestinginhibition at estimated --S--NO concentrations of 150 pM.S-nitroso-proteins synthesized with acidified NaNO₂, with NO gas, or byexposure to bovine aortic endothelial cells stimulated to secrete EDRFwere essentially equipotent, as shown for S-nitroso-BSA in FIG. 10.Furthermore, the platelet inhibitory effect of S-nitroso-BSA (1.4 μM)was confirmed both in platelet-rich plasma and in whole blood (usingimpedance aggregometry in this latter case) (Chong et al., Drug Met. andDisp. 18:61 (1990) herein incorporated by reference).

Representative mean data and illustrative aggregation tracings forS-nitroso-BSA are provided in FIGS. 10 and 11a, respectively.Carboxyamidation of protein thiols with iodoacetamide or pretreatment ofplatelets with the guanylate cyclase inhibitor methylene blue abolishedthe antiplatelet effects of S-nitroso-proteins (FIG. 11a). In addition,the half-life of the antiplatelet effects correlated with that forvascular smooth muscle relaxation.

B. Vasodilation

1. Methods

The vasodilatory actions of S-nitroso-BSA were examined in a standardbioassay containing endothelium-denuded rabbit sonic strips in Kreb'sbuffer, pH 7.5, at 37°, as described in Example 6.

2. Results

Dose-dependent relaxations were observed over the range of 15 nM to 15μM S-nitroso-proteins, and representative mean data for S-nitroso-BSAare provided in FIG. 10. S-nitroso-proteins synthesized with acidifiedNaNO₂, with NO gas, or by exposure to bovine sonic endothelial cellsstimulated to secrete EDRF were essentially equipotent; this is againexemplified for S-nitroso-BSA in FIG. 10. The relaxation response toS-nitroso-BSA proteins differed from that generally ascribed to EDRF,authentic NO, and the relatively labile low molecular weight biologicalS-nitrosothiols, all of which are characterized by rapid, transientrelaxations. In marked contrast, S-nitroso-BSA induced a less rapid, butmuch more persistent, relaxation response (FIG. 11b), thus confirmingthat it acts as a long-acting vasodilator.

Furthermore, BSA incubated with NO synthase in the presence of cofactorsrequired for enzyme activity (calmodulin, NADPH, Ca⁺⁺) showed anL-arginine-dependent ability to induce persistent vasorelaxationcharacteristic of S-nitroso-proteins.

The half-life of S-nitroso-BSA as determined in the bioassaycorresponded with chemical measurements of half-life and isapproximately twenty-four hours. This half-life is significantly longerthan the half-lives of low molecular weight S-nitrosothiols and suggeststhat the temporal profile of the relaxation response for S-nitrosothiolscorrelates with the lability of the S--NO bond.

Blockade of protein thiols by carboxyamidation with iodoacetamideprevented S-nitrosothiol formation as determined chemically, andrendered the proteins exposed to NO or EDRF biologically inactive (FIG.11b). Consonant with the mechanism of other nitro(so)-vasodilators(Ignarro, L. J. Circ. Res. 65:1 (1989)), relaxations were abolished bymethylene blue, an inhibitor of guanylate cyclase (FIG. 11a). Thismechanism was confirmed by showing that S-nitroso-BSA (18 μM) induces3.5-fold increases (n=2) in eyelie GMP over basal levels relative to BSAalone in cultured RFL-6 lung fibroblasts containing a soluble guanylatecyclase exquisitely sensitive to NO (Forstermann et al., Mol. Pharmacol.38:7 (1990)). Stimulation of guanylate cyclase by S-nitroso-BSA wasattenuated by methylene blue.

FIG. 10 demonstrates the dose-dependent relaxation of vascular smoothmuscle and inhibition of platelet aggregation with S-nitroso-BSA(S--NO-BSA). Dose-effect curves for vessel relaxation (▪--▪) andplatelet inhibition (•--•) were generated with S--NO-BSA synthesizedwith equimolar NO generated from acidified NaNO₂ as described in thetext and then neutralized to pH 7.4. Data are presented as mean ±SEM(n=6-18). The open symbols represent experiments, in the vessel (□) andplatelet (◯) bioassays, in which S--NO-BSA was synthesized by exposureof BSA to bovine aortic endothelial cells stimulated to secrete EDRF.These data are presented as mean ±SEM (n=3-8), with the X-axis errorbars indicating the variance in the concentration of S--NO-BSA generatedfrom EDRF and the Y-axis error bars indicating the variance in thebioassay response.

In vessel experiments, relaxations to S--NO-BSA are expressed as percentof tone induced by 1.0 μM norepinephrine.

Infusion of S--NO-BSA into anesthetized dogs, according to standardmethods known in the art, resulted in prolonged decreases in bloodpressure, unmatched by low molecular weight S-nitrosothiols. Inaddition, this compound increased coronary flow, thus preservingmyocardial blood flow. In a canine model of subtotal coronary arteryocclusion, S--NO-BSA inhibited platelet-dependent cyclic thrombusformation and significantly prolonged bleeding times. These extremelypotent, but reversible anti-platelet properties in vivo are unmatched byclassic nitrates. As well, the improvement in coronary blood flownitroprusside markedly with the clinically used nitroso-compound,nitroprusside, which has deleterious effects on coronary flow. As shownin FIGS. 12-14, the constellation of anti-platelet effect, long durationof action, and increased coronary blood flow, is unmatched by othernitroso-compounds. Thus, S-nitroso-proteins have very unique hemodynamicand bioactive profiles.

Example 8: Demonstration of the Vasodilatory Effect ofS-Nitroso-Cathepsin

The effect of S--NO-cathepsin was studied according to the methodsdescribed in Example 7a. Results obtained demonstrated thatS--NO-cathepsin, at a concentration of 150 nM-1.5 μM, inhibits plateletaggregation.

The effect of S--NO-cathepsin on vasodilation was studied according tothe methods described in Example 7b. As shown in the illustrativetracings of FIG. 12, S--NO-cathepsin, at a concentration of 150 nM 1.5μM induces vessel relaxation which is unmatched by equimolar amounts ofnon-nitrosylated cathepsin.

Example 9: Demonstration of the Platelet Inhibitory and VasodilatoryEffect of S-Nitroso-Lipoprotein

The effect of S--NO-LDL on platelet aggregation was studied according tothe methods described in Example 7a. Aggregations were quantified bymeasuring the extent of change of light transmittance, and expressed asa normalized value relative to control aggregations. As shown theillustrative tracings of FIG. 13, inhibition of platelet aggregation isdemonstrable at a concentration of 1 μM S--NO-LDL.

The effect of S--NO-LDL on vasodilation was studied according to themethods described in Example 7b. As shown in FIG. 14, S--NO-LDL inducesvessel relaxation which is unmatched by equimolar mounts ofnon-nitrosylated LDL.

Example 10: Demonstration of the Platelet Inhibitory and VasodilatoryEffect of S-Nitroso-Immunoglobulin

The effect of S--NO-Ig on platelet aggregation was studied according tothe methods described in Example 7a. Aggregations were quantified bymeasuring the extent of change of light transmittance, and expressed asa normalized value relative to control aggregations. As shown in FIG.15, inhibition of platelet aggregation is demonstrable at concentrationsof S--NO-Ig in the pharmacologic range of 150 nM-1.5 μM.

The effect of S--NO-Ig on vasodilation was studied according to themethods described in Example 7b. As shown in FIG. 15, S--NO-Ig, atconcentrations in the range of 150 nM-1.5 μM, induces relaxation whichis unmatched by equimolar amounts of immunoglobulin alone.

Example 11: Relaxation of Airway Smooth Muscle Caused By S-Nitroso-BSA

1. Materials

Glutathione, L-cysteine, DL-homocysteine, D-penicillin, hemoglobinCoovine), methylene blue and Medium 199 sets were purchased from SigmaChemical Co., St. Louis, MO. N-acetylcysteine was obtained from AldrichChemical Co., Milwaukee, WI. Captopril was kindly provided by Dr. VictorDzau. Sodium nitrite, histamine and methacholine were purchased fromFisher Scientific, Fairlawn,. N.J. Leukotriene D₄ was purchased fromAnaquest, BOC Inc., Madison, WI. Antibiotic/antimycotic mixture (10,000U/ml penicillin G sodium, 10,000 mg/ml, streptomycin sulfate, 25 mg/mlamphotericin B) was purchased from Gibco laboratories, Grand Island, NY.Radioimmunoassay kits for the determination of cyclic GMP were purchasedfrom New England Nuclear, Boston, MA.

2. Preparation of Airways

Male Hanley guinea pigs (500-600 g) were anesthetized by inhalation ofenflurance to achieve a surgical plane of anesthesia. The trachea wereexcised and placed in Kreb's-Henseleit buffer (mM); NaCl 118, KCl 5.4,NaH₂ PO₄ 1.01, glucose 11.1, NaHCO₃ 25.0, MgSO₄ 0.69, CaCl 2.32, pH 7.4.The airways were then dissected free from surrounding fat and connectivetissue and cut into tings 2-4 mm in diameter. The trachea rings wereplaced in sterile Medium 199 containing 1% antibiotic/antimycoticmixture in an atmosphere of 5% CO₂, 45% O₂, 55% N₂ and kept for up to 48hours in tissue culture. The experiments were also performed on humanairways isolated by the same method.

3. Bioassay

Trachea tings were mounted on stirrups and connected to transducers(model FTO3C Grass), by which change-in isometric tension were measured.Rings were then suspended in 10 cc of oxygenated (95% O2, 5% CO₂)buffer. Airway rings were equilibrated for 60 minutes under a load of 1gm and then primed twice by exposure to 100 μM methacholine. The ringswere contracted with various agonists at concentrations determined togenerate 50% (±16% S.D.) of maximum tone, after which the effect ofS--NO-BSA was assessed. In selected experiments, relaxation responseswere determined in the presence of hemoglobin, or after rings had beenpreexposed to methylene blue for 30 minutes.

4. Results

As shown in FIG. 17, S--NO-BSA is a potent airway smooth musclerelaxant, producing 50% relaxation at a concentration of 0.01 μM andover 75% relaxation at a concentration of 10 μM.

Example 12: Inhibition of Enzymatic Activity of Cathepsin B byNitrosylation

The enzymatic activity of S--NO-cathepsin B was measured against thechromogenic substrate, S2251 at pH 5, in sodium acetate buffer.S-nitrosylation resulted in a loss of enzymatic activity.

Example 13: Nitrosylation of Aromatic Amino Acids

1. Methods

a. Preparation of Nitroso-tyrosine

50 mmol of L-tyrosine (Sigma Chemical company; St. Louis, MO) weredissolved into 0.5 ml of distilled water. 250 mmol of Na¹⁵ NO₂ (sodiumN-[15] nitrite: MSD Isotopes, Merck Scientific; Rahway, NJ) weredissolved into 0.5 mL of 1N HCL (Fisher Scientific; Fair Lawn, NJ) andtransferred immediately to the aqueous tyrosine solution with agitationby Vortex stirrer. Solution was capped and allowed to sit at roomtemperature for 30 minutes. NMR measurements were made as follows:

(a) ¹⁵ N-NMR: D₂ O was added and measurements were taken immediately;

(b) ¹ H-NMR: After ¹⁵ N-NMR was completed, solution was removed andplaced into a small round-bottom flask and water was removed in vacuo.D₂ O was added to the dry off-white solid (this time as a solvent) andmeasurements were run immediately;

(c) Infrared Spectroscopy: Fourier Transform Infrared Spectroscopy(FFIR) samples were prepared through removal of water (as in b)) andsubsequent creation of a Nujol Mull using mineral oil.

(d) ultraviolet and Visible Spectroscopy (UV-Vis): Samples for UV-Visexamination were used as per above prep without further modification.Samples were referenced to distilled water.

b. Nitrosylation of Phenylalanine, Tyrosine, and L-Boc-Tyr (Et)-OH.

50 mmol of L-phenylalanine, L-tyrosine (Sigma Chemical Company; St.Louis, MO), or L-boc-tyr(Et)-OH (Bachera Bioscientific Incorporated;Philadelphia, PA) were dissolved into 0.5 ml of distilled water. 250mmol of NA¹⁵ NO₂ (sodium N-[15] nitrite) were dissolved into 0.5 ml of1N HCl (aq). and transferred immediately to the aqueous amino acidsolution with agitation by Vortex stirrer. Solution was capped andallowed to sit at room temperature for 30 minutes. ¹⁵ N-NMR and ¹ H-NMRwere performed as per nitroso-tyrosine above. Standard reference oftyrosine for FTIR was prepared as a Nujol Mull of pure crystallineL-tyrosine.

c. Nitrosylation of Tryptophan

1.7 mM of tryptophan were reacted with equimolar NaNO₂ in 0.5N HCl fortime periods of 5, 10, 15 and 60 minutes at 25° C.

2. Results

a. 15N-NMR data

All NMR [¹⁵ N and ¹ H] were run on two Bruker AM-500 MgHz spectrometers.Nitrosylation of tyrosine at pH 0.3 gives signals at approximately 730ppm and 630 ppm relative to saturated sodium N-[15]nitrite aqueoussolution referenced at 587 ppm¹² (¹⁵ NO₂) (FIG. 21a.) A signal at 353ppm (aqueous NO¹²) was also observed. Nitrosylation of phenylalanineunder the same conditions gave the signal at approximately 630 ppm butnot the 730 ppm signal despite repeated attempts (FIG. 22).Nitrosylation of phenylalanine also yielded signals at 587 ppm (excess,unprotonated nitrite) and 353 ppm. Nitrosylation of O-blocked tyrosinemodel, boc-tyr(Et)-OH, also yielded a signal at approximately 630 ppm;and others at 587 ppm and 353 ppm. Small signals in the range 450-495ppm were observed for the tyrosine models, phe and boc-Wr(Et)-OH.

b. 1H-NMR data

To further characterize the nitrosylation of the phenolic functionalityof L-tyr to the exclusion of C-nitrosylation, proton-NMR was performedon nitrosylated tyrosine; modification of L-tyr at the phenolic-OH wouldnot appear in proton-NMR because of proton exchange with the deuteratedsolvent (D₂ O). Examination of the spectra showed the classic doublet ofdoublets at low field, which is characteristic of para-disubstitutedbenzene, thus excluding aromatic proton substitution (FIG. 21b). This,and other values in the spectra were characteristic of unmodified L-tyr.

c. FTIR data

All FTIR were run on a Nicolet 5ZDX FT-IR Spectrometer. FTIR of a NujolMull of L-tyrosine showed a very characteristic and well-documentedalcoholic stretch in the spectra due to the phenolic-OH (FIG. 1d.inlaid). This spectrum lacked any signal(s) at the 1680-1610 cm⁻¹ rangethat coincides with the O--N═O stretch (not shown). FTIR of nitrosylatedL-tyrosine showed no evidence of alcoholic--OH stretches and containedtwo small bands in the range of 1680-1610 cm⁻¹ that could possiblyaccount for the expected O--M═O stretch (Wade, L. G., Organic Chemistry(1st Ed.) Prentice-Hall Inc., Englewood Cliffs, N.J.: 1987. p. 1334)(FIG. 21c.).

d. UV-Vis data

All UV-Vis spectroscopy was performed using a Gilford Response UV-VisSpectrophotometer (CIBA-Coming, Oberlin; OH). Treatment of L-tyrosinewith aqueous sodium nitrite at pH 0.3 (0.5N HCl) resulted in a yellowsolution with an absorption maximum at 361 nm. This result is similarto, but differs from previously reported results with nitrosatedL-tyrosine. Ortho-ring substituted L-nitro-tyrosine (Sigma) absorbs at356 nm at pH 0.3.

Treatment of phenylalanine with sodium nitrite at pH 0.3 gives a rapidlychanging UV spectrum with a peak increasing in wavelength from 318 nm at5 min. to a maximum unchanging peak at 527 nm by 30 min.

FIG. 23(a-e) demonstrates time-dependent nitrosylation of tryptophan.The data is suggestive of nitrosylation of both the aromatic ring andamino groups.

Example 14: Nitrosylation of BSA

BSA, at 200 mg/ml, was loaded at a ratio of 20:1 with NO in 0.5N HCl for30 minutes at room temperature. As shown in FIG. 24, the 726 ppm peakindicates O-nitrosation of the tyrosine residues on BSA. FIG. 24 aimprovides evidence for the nitrosation of several other functional groupson BSA. The data are also suggestive of ring nitrosation and aminenitrosation (600 ppm peak) as well.

Time-dependent NO loading of BSA was performed by exposing BSA (200mg/ml) in phosphate buffer (10 mM, pH 7.4) to NO gas bubbled into theBSA solution, for 1, 5 and 30 minute time periods. FIG. 25 provides UVspectrum data which indicates NO loading of BSA.

Example 15: Nitrosylation of t-PA; NO Loading

t-PA at 10 mg/ml was exposed 10:1 to excess NaNO₂ in 0.5N HCl. FIG. 26shows NO-loading of t-PA.

Example 16. Vasodilatory Effect of NO-Loaded BSA

BSA was loaded with NO according to the method described in Example 14.Vasodilatory effect was studied in a rabbit aorta bioassay, according tothe methods described in Example 6C. As shown in FIG. 27, increasingconcentrations of NO resulted in an increase in vessel relaxationinduced by the resultant NO-BSA.

Example 17. Guanylate Cyclase Inhibitors Do Not InhibitS-nitroso-protein-Induced Relaxation in Human Airways

The effect of guanylate cyclase inhibitors uponS-nitroso-protein-induced airway relaxation and cGMP increase wasassessed, using the previously described bioassay and cyclic nucleotideassay procedures. The bronchodilatory effect of S-nitroso-albumin wasexamined in human airways (5-12 mm outer diameter).Concentration-response relationships for rings contracted withmethacholine (7 μM) resulted in IC50 values of 22 μM, approximately twoorders of magnitude greater than theophylline.

S-nitroso-albumin (100 μM) induced increases over control airway cGMPlevels. However, S-nitroso-albumin-induced airway relaxation was notsignificantly inhibited by methylene blue (10⁻⁴) or LY83583 (5×10⁻⁵).Similarly, hemoglobin (100 μM) had little effect onS-nitroso-albumin-induced relaxation (P═NS).

These results demonstrate that the mechanism by which S-nitroso-proteincause airway relaxation is not due solely to increases in cGMP. Thus,S-nitroso-proteins cause airway relaxation through both an increase incyclic GMP, as well as a cGMP-independent pathway. In doing so, theyprovide a means for achieving combination therapy by maximizing thesynergistic effect of two separate mechanisms.

Example 18: S-nitroso-proteins resist Decomposition in the Presence ofRedox Metals

The stability of S-nitroso-albumin in the presence of oxygen and redoxmetals was assessed. When subjected to conditions consisting of 95% O₂,pH 7.4, the half life of this compound was shown to be on the order ofhours, and significantly greater than that of NO, or NO•, which, undersimilar conditions, are on the order of seconds.

In addition, S-nitroso-protein stability was assessed in the presence ofvarious redox metals or chelating agents. S-nitroso-albumin wasresistant to decomposition when Cu⁺, Fe²⁺, or Cu²⁺ (50 μM) ordefuroxamine or EDTA (10 μM) were added. Thus, these experimentsdemonstrate that, unlike NO•, S-nitroso-proteins are not rapidlyinactivated in the presence of oxygen, nor do they decompose in thepresence of redox metals.

Example 19. S-nitrosylation of Hemoglobin Increases Hemoglobin-oxygenBinding

Additional experiments were conducted to evaluate the reaction betweenS-nitrosothiols and hemoglobin. S-nitrosylation of hemoglobin wasaccomplished by reacting 12.5 μM hemoglobin with 12.5 μM for 5 and 20minute intervals (pH 6.9). S-nitrosylation was verified, using standardmethods for detection of S-nitrosothiols (Saville, Analyst 83:670-672(1958)). The Saville method, which assays free NO_(x) in solution,involves a diazotization reaction with sulfanilamide and subsequentcoupling with the chromophore N-(1-naphthyl)ethylenediamine. Thespecificity for S-nitrosothiols derives from assay determinationsperformed in the presence and absence of HgCl₂, the latter reagentcatalyzing the hydrolysis of the S--NO bond. Confirmatory evidence forS-nitrosothiol bond formation was obtained by spectrophotometry,demonstrated by the absorption maximum of 450 nm, as shown in FIG. 28.This was demonstrated using NO⁺ equivalents in the form of SNOAC.

As demonstrated by FIG. 29, the UV spectrum of hemoglobin incubated withSNOAC shows no reaction at the redox metal (iron-binding site) ofhemoglobin, over 15 minutes. For the purposes of comparison, equimolarconcentrations of hemoglobin and NaNO₂ were reacted in 0.5N HCl, to formnitrosyl-hemoglobin, and the UV spectrum was obtained. As shown in FIG.30, NO reacted instantaneously with the redox metal site on hemoglobin.The fact that the S-nitrosothiol did not react with the redox metal siteof hemoglobin, but with its thiol group instead, indicates that thereactive NO species donated by the S-nitrosothiol is nitrosonium ornitroxyl.

S-nitrosylation of hemoglobin does not result in the formation ofmethemoglobin and consequent impairment in hemoglobin-oxygen binding.Furthermore, an additional experiment demonstrated that S-nitrosylationof hemoglobin causes a leftward shift in the hemoglobin-oxygenassociation curve, indicating an increase in oxygen binding. Thus, thereaction between S-nitrosothiols and hemoglobin not only eliminates theinhibition of oxygen binding which occurs from the reaction withuncharged NO and generation of methemoglobin, but it actually increasesoxygen binding.

Having now fully described this invention, it will be appreciated bythose skilled in the an that the same can be performed within a widerange of equivalent parameters, concentrations, and conditions withoutdeparting from the spirit and scope of the invention and without undueexperimentation.

While this invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications. This application is intended to cover any variations,uses, or adaptations of the inventions following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as my be applied to theessential features hereinbefore set forth as follows in the scope of theappended claims.

What is claimed is:
 1. A nitrosylated thrombolytic polypeptide selectedfrom the group consisting of a nitrosylated tissue-type plasminogenactivators, nitrosylated streptokinase and nitrosylated urokinaseproduced by a method selected from the group consisting of:(a) exposingthe polypeptide to a nitric oxide donor compound under conditions whichpermit the release or transfer from said donor and the binding ortransfer to said polypeptide of at least one nitric oxide moiety: (b)passing a gaseous source of nitric oxide through a solution of thepolypeptide to an extent sufficient to effect the binding or transfer tosaid polypeptide of at least one nitric oxide moiety; (c) incubating thepolypeptide with cells which have been stimulated to secrete EDRF to anextent sufficient to effect the binding or transfer to said polypeptideof at least one nitric oxide moiety; and (d) incubating the polypeptidewith nitric oxide synthase, and a substrate and cofactor therefor, to anextent sufficient to effect the binding to said polypeptide of at leastone nitric oxide moiety.
 2. The nitrosylated thrombolytic polypeptide ofclaim 1 which is produced by nitrosylating a thrombolytic polypeptidewith an equimolar amount of a compound that transfers nitrogen monoxidethereto.
 3. The nitrosylated thrombolytic polypeptide of claim 1 whichis produced by nitrosylating a tissue-type plasminogen activator with acompound selected from the group consisting of NaNO₂, NOCl, N₂ O₃, N₂ O₄and NO⁺.
 4. The nitrosylated thrombolytic polypeptide of claim 1 whichis produced by nitrosylating an aqueous solution of the thrombolyticpolypeptide with nitric oxide gas.
 5. The nitrosylated thrombolyticpolypeptide of claim 1 which is a nitrosylated tissue-type plasminogenactivator produced by nitrosylating a tissue-type plasminogen activator.6. The nitrosylated tissue-type plasminogen activator of claim 5 whichis produced by nitrosylating a tissue-type plasminogen activator with anequimolar amount of a compound that transfers nitrogen monoxide thereto.7. The nitrosylated tissue-type plasminogen activator of claim 5 whichis produced by nitrosylating a tissue-type plasminogen activator with acompound selected from the group consisting of NaNO₂, NOCl, N₂ O₃, N₂ O₄and NO⁺.
 8. The nitrosylated tissue-type plasminogen activator of claim5 which is produced by nitrosylating an aqueous solution of atissue-type plasminogen activator with nitric oxide gas.
 9. Thenitrosylated thrombolytic polypeptide of claim 1 which is a nitrosylatedstreptokinase produced by nitrosylating a streptokinase.
 10. Thenitrosylated streptokinase of claim 9 which is produced by nitrosylatinga streptokinase with an equimolar amount of a compound that transfersnitrogen monoxide thereto.
 11. The nitrosylated streptokinase of claim 9which is produced by nitrosylating a streptokinase with a compoundselected from the group consisting of NaNO₂, NOCl, N₂ O₃, N₂ O₄ and NO⁺.12. The nitrosylated streptokinase of claim 9 which is produced bynitrosylating an aqueous solution of a streptokinase with nitric oxidegas.
 13. The nitrosylated thrombolytic polypeptide of claim 1 which is anitrosylated urokinase produced by nitrosylating a urokinase.
 14. Thenitrosylated urokinase of claim 13 which is produced by nitrosylating aurokinase with an equimolar amount of a compound that transfers nitrogenmonoxide thereto.
 15. The nitrosylated urokinase of claim 13 which isproduced by nitrosylating a urokinase with a compound selected from thegroup consisting of NaNO₂, NOCl, N₂ O₃, N₂ O₄ and NO⁺.
 16. Thenitrosylated urokinase of claim 9 which is produced by nitrosylating anaqueous solution of a urokinase with nitric oxide gas.